The invention pertains to the co-culture of hematopoietic progenitor cells and lymphoreticular stromal cells in three-dimensional devices, resulting in unexpectedly high numbers of lymphoid tissue-specific cell progeny.
A characteristic of the immune system is the specific recognition of antigens. This includes the ability to discriminate between self and non-self antigens and a memory-like potential that enables a fast and specific reaction to previously encountered antigens. The vertebrate immune system reacts to foreign antigens with a cascade of molecular and cellular events that ultimately results in the humoral and cell-mediated immune response.
The major pathway of the immune defense involving antigen-specific recognition commences with the trapping of the antigen by antigen presenting cells (APCs), such as dendritic cells or macrophages, and the subsequent migration of these cells to lymphoid organs (e.g., thymus). There, the APCs present antigen to subclasses of T cells classified as mature T helper cells. Upon specific recognition of the presented antigen, the mature T helper cells can be triggered to become activated T helper cells. The activated T helper cells regulate both the humoral immune response by inducing the differentiation of mature B cells to antibody producing plasma cells and the cell-mediated immune response by activation of mature cytotoxic T cells.
The thymus has been shown to be an obligatory factor in T cell differentiation of hematopoietic cells. Based upon the murine model, it is believed that the presence of a three dimensional organ is required, as in vitro models that do not include the thymus and a three dimensional structure fail to support T cell lymphopoiesis (Owen J J, et al., Br Med Bull., 1989, 45:350-360). The process of differentiation, however, appears to begin prior to progenitor cells contacting the thymus.
Primitive hematopoietic progenitors in the fetal liver or bone marrow give rise to lineage committed cells, including progenitors committed to the T lymphoid lineage. These most immature cells are identified by the surface expression of CD34. T cell lineage committed cells express CD34, but no discrete expression of other epitopes found only on T cell progenitors has been described. Further, T lymphocyte differentiation normally occurs via a series of discrete developmental stages. Primitive progenitor cells which do not express lymphocyte specific cell surface markers (CD34+ CD3xe2x88x92 CD4xe2x88x92 CD8xe2x88x92) migrate to the thymus where they acquire, through a series of maturational events, the phenotype CD34xe2x88x92 CD3xe2x88x92 CD4+ CD8xe2x88x92. These cells then mature into double positive CD4+ CD8+ cells, most of which are CD3+, although CD3 expression is not universally detectable. These cells further undergo both positive and negative selection, and mature to develop into single positive T cells (CD4+ CD8xe2x88x92 or CD4xe2x88x92 CD8+). These cells ultimately migrate into the peripheral circulation as naive T cells.
T cell disorders and diseases represent major health problems. Recent progress has been made using gene therapy to treat conditions involving T lymphocytes, including AIDS. This has fostered increased interest in the development of laboratory techniques that allow in vitro evaluations of potential genetic therapies for these conditions.
The understanding of T cell differentiation has been hampered by the limited availability of technologies which permit in vitro T cell differentiation. To date, T cell differentiation studies have been largely confined to the SCID-hu mouse in vivo model. In vitro technologies have been based on thymic explant studies and primate thymic monolayers. In a recent advance, primate thymic stroma cultures have been shown to provide an expedient, although inefficient, system for examining T cell development, enabling in vitro T cell differentiation in a reproducible manner. However, the purity and number of T cells generated this way, as well as the relatively short half-life of the cultures, generally results in limited applicability to more advanced studies of T cell differentiation and function.
The invention, in one important part, involves improved methods for culturing hematopoietic progenitor cells that direct their development toward lymphoid tissue-specific lineages without the addition of exogenous growth factors. Thus, one aspect of the invention is the culture of hematopoietic progenitor cells to generate progeny committed to a specific lineage. Another aspect is an improvement in the rate and the number of differentiated progeny that can be obtained from a sample of hematopoietic progenitor cells.
We describe herein a system that takes advantage of biocompatible, open-pore, three-dimensional matrices, and uses human and non-human lymphoreticular stromal cells to provide the appropriate conditions for the expansion and differentiation of human and non-human hematopoietic progenitor cells toward a specific cell lineage. T lymphocytes, for example, derived from these cultures respond normally to a variety of stimuli and express the diversity of markers expected of mature T cells.
This system provides significant advantages over existing techniques. For example, it can provide for the rapid generation of a large number of differentiated progeny necessary for laboratory analysis and/or therapeutic uses, including for in vitro testing of potential gene therapy strategies or for reinfusion into subjects in vivo. The matrix itself can be implanted into subjects for in vivo studies of hematopoietic cell growth. The system also can reasonably replicate the complex process of hematopoietic cell maintenance, expansion and/or differentiation toward a specific lineage.
Surprisingly, according to the invention, it has been discovered that hematopoietic progenitor cells co-cultured with lymphoreticular stromal cells in a porous solid scaffold, without the addition of exogenous growth agents, generate at a fast rate an unexpectedly high number of functional, differentiated progeny of a lymphoid-specific lineage. The lymphoid tissue from which lymphoreticular stromal cells are derived helps determine the lineage-commitment hematopoietic progenitor cells undertake, resulting in the lineage-specificity of the differentiated progeny. Also surprising, according to the invention, is the discovery that lesser amounts of nonlymphoid cells (i.e. myelo-monocytic cells) are generated from the co-culture of hematopoietic progenitor cells and lymphoreticular stromal cells in a porous solid scaffold of the invention when compared to existing methodology. Thus, the present invention permits for the rapid generation of a large number of differentiated, lymphoid-specific cells from a relatively small number of hematopoietic progenitor cells. Such results were never before realized using known art methodologies (e.g., as in U.S. Pat. No. 5,677,139 by Johnson et al., which describes the in vitro differentiation of CD3+ cells on primate thymic stroma monolayers, or as in U.S. Pat. No. 5,541,107 by Naughton et al., which describes a three-dimensional bone marrow cell and tissue culture system).
According to one aspect of the invention, a method for in vitro production of lymphoid tissue-specific cells is provided. The method involves introducing an amount of hematopoietic progenitor cells and an amount of lymphoreticular stromal cells into a porous, solid matrix having interconnected pores of a pore size sufficient to permit the hematopoietic progenitor cells and the lymphoreticular stromal cells to grow throughout the matrix. The hematopoietic progenitor cells and the lymphoreticular stromal cells are then co-cultured. The amount of the lymphoreticular stromal cells utilized is sufficient to support the growth and differentiation of the hematopoietic progenitor cells. In one embodiment, co-culturing occurs under conditions sufficient to produce at least a 10-fold increase in the number of lymphoid tissue-specific cells. In preferred embodiments, co-culturing occurs under conditions sufficient to produce at least a 20, 50, 100, 200, 300 or 400-fold increase in the number of lymphoid tissue-specific cells. In some embodiments, after the co-culturing, harvesting of the lymphoid tissue-specific cells may be performed.
In certain embodiments, the hematopoietic progenitor cells may be pluripotent stem cells, multipotent progenitor cells and/or progenitor cells committed to specific hematopoietic lineages. The progenitor cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage.
The hematopoietic progenitor cells may be derived from a tissue such as bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue. Lymphoid soft tissue includes the thymus, spleen, liver, lymph node, skin, tonsil and Peyer""s patches. In other embodiments, the lymphoreticular stromal cells may be also derived from at least one of the foregoing lymphoid soft tissues. In important embodiments, the lymphoreticular stromal cells are thymic stromal cells and the multipotent progenitor cells and/or committed progenitor cells are committed to a T cell lineage. In further important embodiments, the lymphoreticular stromal cells are skin-derived stromal cells and the multipotent progenitor cells and/or committed progenitor cells are committed to a T cell lineage. In other embodiments, the hematopoietic progenitor cells and/or the lymphoreticular stromal cells may be genetically altered.
In certain embodiments, the hematopoietic progenitor cells and the lymphoreticular stromal cells are autologous (e.g., originate from the same individual). In important embodiments, the method further comprises antigen presenting cells. In some embodiments, the hematopoietic progenitor cells, the lymphoreticular stromal cells, and the antigen presenting cells are all autologous. In one embodiment, the method further comprises antigen presenting cells non-autologous to the hematopoietic progenitor cells and the lymphoreticular stromal cells.
In some embodiments, the hematopoietic progenitor cells and the lymphoreticular stromal cells are non-autologous (e.g., allogeneic, syngeneic and/or xenogeneic in origin). In important embodiments, the method further comprises antigen presenting cells. Various embodiments are provided wherein different source combinations for each of the cells in the co-culture are encompassed by the present invention. For example, the hematopoietic progenitor cells, the lymphoreticular stromal cells, and the antigen presenting cells can be autologous, or non-autologous and each one from a different source. In another instance, the hematopoietic progenitor cells and the antigen presenting cells can be autologous or non-autologous. In still another instance, the lymphoreticular stromal cells and the antigen presenting cells are non-autologous.
In certain embodiments, antigen presenting cells may be added to the co-culture of hematopoietic progenitor cells and lymphoreticular stromal cells. Various embodiments are provided encompassing different source combinations for each of the cells in the co-culture, and wherein the lymphoid tissue-specific cells produced are to be used in transplantation into a host. For example, each of the hematopoietic progenitor cells, lymphoreticular stromal cells and/or antigen presenting cells may be autologous or non-autologous to the cells of the host.
In any of the foregoing aspects and embodiments of the invention at least one antigen may be included, or added after, the co-culture of the cells. In any of the foregoing embodiments involving antigen presenting cells, it is preferred that the antigen presenting cells are mature.
According to any of the foregoing aspects and embodiments, the method of the invention can include hematopoietic progenitor cells, lymphoreticular stromal cells and/or antigen presenting cells that are genetically altered.
In one important embodiment of the invention, the hematopoietic progenitor cells are of human origin and the lymphoreticular stromal cells are also of human origin. Antigen presenting cells can also be of human and non-human origin. In another embodiment, the hematopoietic progenitor cells are of human origin and the lymphoreticular stromal cells are of non-human origin. In preferred embodiments, non-human lymphoreticular stromal cells are of murine origin.
In certain embodiments, the lymphoreticular stromal cells are seeded to the matrix at the same time as the hematopoietic progenitor cells. In other embodiments, the lymphoreticular stromal cells are seeded to the matrix prior to inoculating the hematopoietic progenitor cells.
The porous matrix can be one that is an open cell porous matrix having a percent open space of at least 50%, and preferably at least 75%. In one embodiment the porous solid matrix has pores defined by interconnecting ligaments having a diameter at midpoint, on average, of less than 150 xcexcm. Preferably the porous solid matrix is a metal-coated reticulated open cell foam of carbon containing material, the metal coating being selected from the group consisting of tantalum, titanium, platinum (including other metals of the platinum group), niobium, hafnium, tungsten, and combinations thereof. In preferred embodiments, whether the porous solid matrix is metal-coated or not, the matrix is coated with a biological agent selected from the group consisting of collagens, fibronectins, laminins, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, functional equivalents of these factors (including fragments thereof), and combinations thereof. Most preferably the metal coating is tantalum coated with a biological agent. In certain other embodiments, the porous solid matrix having seeded hematopoietic progenitor cells and their progeny, and lymphoreticular stromal cells (and/or antigen presenting cells), is impregnated with a gelatinous agent that occupies pores of the matrix.
The preferred embodiments of the invention are solid, unitary macrostructures, i.e. not beads or packed beads. They also involve nonbiodegradable materials.
According to any of the foregoing embodiments, the method of the invention can include culturing the cells in an environment that is free of hematopoietic progenitor cell survival and proliferation factors such as interleukins 3, 6 and 11. Still another embodiment of the invention is performing the co-culturing of the hematopoietic progenitor cells and the lymphoreticular stromal cells in an environment that is free altogether of stromal cell conditioned medium and exogenously added hematopoietic growth factors that promote hematopoietic cell maintenance, expansion and/or differentiation, other than serum.
As will be understood, according to the invention, it is possible now to co-culture hematopoietic progenitor cells and lymphoreticular stromal cells (that may or may not include antigen presenting cells), in an environment that is free of exogenously added hematopoietic growth factors that promote hematopoietic cell maintenance, expansion and/or differentiation for as little as 7 days and to obtain large numbers of differentiated progeny of a specific lineage.
According to any of the foregoing embodiments, the method of the invention can include co-culturing of the hematopoietic progenitor cells, the lymphoreticular stromal cells, and/or the antigen presenting cells with an exogenously added agent selected from the group consisting of stromal cell conditioned medium, and a hematopoietic growth factor that promotes hematopoietic cell maintenance, expansion and/or differentiation, and influences cell localization. In certain embodiments, the hematopoietic growth factor that promotes hematopoietic cell maintenance, expansion and/or differentiation, and influences cell localization, may be an agent that includes interleukin 3, interleukin 6, interleukin 7, interleukin 11, interleukin 12, stem cell factor, FLK-2 ligand, FLT-2 ligand, Epo, Tpo, GMCSF, GCSF, Oncostatin M, and MCSF.
According to another aspect of the invention, a method for in vivo maintenance, expansion and/or differentiation of hematopoietic progenitor cells is provided. The method involves implanting into a subject a porous, solid matrix having seeded therein hematopoietic progenitor cells (which may include their progeny) and lymphoreticular stromal cells. The porous matrix has interconnected pores of a pore size sufficient to permit the cells to grow throughout the matrix and is an open cell porous matrix having a percent open space of at least 50%, and preferably at least 75%. Various embodiments are provided, wherein the porous solid matrix has one or more of the preferred characteristics as described above.
In certain embodiments, hematopoietic progenitor cells (that may include progeny) and lymphoreticular stromal cells are attached to the matrix by introducing in vitro an amount of hematopoietic progenitor cells and an amount of lymphoreticular stromal cells into the porous solid matrix, and co-culturing the hematopoietic progenitor cells in an environment that is free of stromal cell conditioned medium and free of exogenously added hematopoietic growth factors that promote hematopoietic cell maintenance, expansion and/or differentiation, other than serum. Various other embodiments are provided, wherein the co-culturing is performed under conditions as described above. In yet other embodiments, the porous solid matrix having seeded hematopoietic progenitor cells (that may include progeny) and lymphoreticular stromal cells is impregnated with a gelatinous agent that occupies pores of the matrix.
According to one aspect of the invention, a method for inducing T cell tolerance, is provided. The method involves producing lymphoid tissue-specific cells according to any of the foregoing co-culture methods of the invention that involve the co-culture of cells that include non-autologous cells, under conditions sufficient to induce the formation of T cells and/or T cell progenitors and to inhibit immune activation of the formed cells.
According to yet another aspect of the invention, a method for treating a subject to enhance immune tolerance in the subject, is provided. The method involves administering to a subject in need of such treatment an amount of lymphoid tissue-specific cells produced according to any of the foregoing co-culture methods of the invention that involve the co-culture of cells that may include non-autologous cells, wherein the amount of lymphoid tissue-specific cells is sufficient to enhance in the subject immune tolerance to an autologous or a non-autologous antigen. Various embodiments are provided wherein preferred cell types and porous matrix are as described elsewhere herein (see, e.g., below).
According to still another aspect of the invention, a method for inducing T cell reactivity, is provided. The method involves producing lymphoid tissue-specific cells according to any of the foregoing co-culture methods of the invention that involve the co-culture of cells that may include autologous and/or non-autologous cells, in the presence of at least one antigen, under conditions sufficient to induce formation of T cells or T cell progenitors having specificity for the at least one antigen. In important embodiments, the at least one antigen is added to the co-culture in a further step after formation of T cells or T cell progenitors.
In certain embodiments, the hematopoietic progenitor cells may be pluripotent stem cells, multipotent progenitor cells and/or progenitor cells committed to specific hematopoietic lineages.
The hematopoietic progenitor cells may be derived from a tissue such as bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue. Lymphoid soft tissue includes the thymus, spleen, liver, lymph node, skin, tonsil and/or Peyer""s patches. In other embodiments, the lymphoreticular stromal cells may be also derived from at least one of the foregoing lymphoid soft tissues. In preferred embodiments, the lymphoreticular stromal cells are thymic stromal cells and the multipotent progenitor cells and/or committed progenitor cells are committed to a T cell lineage. In other embodiments, the hematopoietic progenitor cells and/or the lymphoreticular stromal cells may be genetically altered.
In important embodiments, antigen presenting cells may be added to the co-culture. Antigen presenting cells include cells such as dendritic cells, monocytes/macrophages, Langerhans cells, Kupfer cells, microglia, alveolar macrophages and B cells. In other embodiments, the antigen presenting cells are derived from hematopoietic progenitor cells in vitro. Various embodiments are provided, wherein the hematopoietic progenitor cells, the lymphoreticular stromal cells, and the porous solid matrix have one or more of the preferred characteristics as described above, and the cells are cultured as described above. The antigen presenting cells may be derived from hematopoietic progenitor cells in vitro. In important embodiments the antigen presenting cells are mature. In further embodiments, the method further comprises administering a co-stimulatory agent to the co-culture. Preferred co-stimulatory agents include lymphocyte function associated antigen 3 (LFA-3), CD2, CD40, CD80/B7-1, CD86/B7-2, OX-2, CD70, and CD82.
In yet another aspect of the invention, a solid porous matrix is provided wherein hematopoietic progenitor cells, with or without their progeny, and lymphoreticular stromal cells are attached to the solid porous matrix. The lymphoreticular stromal cells are present in an amount sufficient to support the growth and differentiation of hematopoietic progenitor cells. In certain embodiments, the hematopoietic progenitor cells are attached to the lymphoreticular stromal cells. In further embodiments, the solid porous matrix may include antigen presenting cells (progeny and/or nonprogeny). Preferably the antigen presenting cells are mature. In yet further embodiments, the porous matrix further comprises at least one antigen. The porous matrix can be one that is an open cell porous matrix having a percent open space of at least 50%, and preferably at least 75%. In one embodiment the porous solid matrix has pores defined by interconnecting ligaments having a diameter at midpoint, on average, of less than 150 xcexcm. Preferably the porous solid matrix is a metal-coated reticulated open cell foam of carbon containing material, the metal coating being selected from the group consisting of tantalum, titanium, platinum (including other metals of the platinum group), niobium, hafnium, tungsten, and combinations thereof. In preferred embodiments, whether the porous solid matrix is metal-coated or not, the matrix is coated with a biological agent selected from the group consisting of collagens, fibronectins, laminins, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, functional equivalents of these factors, and combinations thereof. Most preferably the metal coating is tantalum coated with a biological agent. In certain other embodiments the porous solid matrix having seeded hematopoietic progenitor cells and lymphoreticular stromal cells, is impregnated with a gelatinous agent that occupies pores of the matrix.
In a further aspect of the invention, a method for identifying an agent suspected of affecting hematopoietic cell development, is provided. The method involves introducing an amount of hematopoietic progenitor cells and an amount of lymphoreticular stromal cells into a porous, solid matrix having interconnected pores of a pore size sufficient to permit the hematopoietic progenitor cells and the lymphoreticular stromal cells to grow throughout the matrix, co-culturing the hematopoietic progenitor cells and the lymphoreticular stromal cells in the presence of at least one candidate agent suspected of affecting hematopoietic cell development (in a test co-culture), and determining whether the at least one candidate agent affects hematopoietic cell development in the test co-culture by comparing the test co-culture hematopoietic cell development to a control co-culture, whereby hematopoietic progenitor cells and lymphoreticular stromal cells are co-cultured in the absence of the at least one candidate agent. Various embodiments are provided, wherein the hematopoietic progenitor cells, the lymphoreticular stromal cells, and the porous solid matrix have one or more of the preferred characteristics as described above, and the cells are cultured as described above. In certain embodiments, hematopoietic progenitor cell development includes hematopoietic progenitor cell maintenance, expansion, differentiation toward a specific cell lineage, and/or cell-death (including apoptosis). In preferred embodiments the lymphoreticular stromal cells are thymic stromal cells.
In another aspect of the invention, a method for isolating from a cell culture an agent suspected of affecting hematopoietic cell development, is provided. The method involves introducing an amount of hematopoietic progenitor cells and an amount of lymphoreticular stromal cells into a porous, solid matrix having interconnected pores of a pore size sufficient to permit the hematopoietic progenitor cells and the lymphoreticular stromal cells to grow throughout the matrix, co-culturing the hematopoietic progenitor cells and the lymphoreticular stromal cells, obtaining a test-supernatant from the co-culture, comparing the test-supernatant to a control-supernatant, and obtaining a subfraction of the test-supernatant that contains an agent suspected of affecting hematopoietic cell development that is absent from the control-supernatant. In certain embodiments the agent suspected of affecting hematopoietic cell development may be present in the control-supernatant and absent from the test-supernatant. In other embodiments, the agent suspected of affecting hematopoietic cell development in one supernatant may be different to an agent suspected of affecting hematopoietic cell development in the other supernatant (e.g., in size, via a post-translational modification, in an alternatively spliced variant form, etc.). Various embodiments are provided, wherein the hematopoietic progenitor cells, the lymphoreticular stromal cells, and the porous solid matrix have one or more of the preferred characteristics as described above, and the cells are cultured as described above. In certain embodiments, hematopoietic progenitor cell development includes hematopoietic progenitor cell maintenance, expansion, differentiation toward a specific cell lineage, and/or cell-death (including apoptosis). In preferred embodiments, the lymphoreticular stromal cells are thymic stromal cells. In certain other embodiments, the control culture system of the prior art (where the control-supernatant can be obtained from) is the one described in U.S. Pat. No. 5,677,139 by Johnson et al.
These and other aspects of the invention, as well as various advantages and utilities, will be more apparent with reference to the detailed description of the preferred embodiments.